Dong Hua

Essential role for TrpC5-containing extracellular vesicles in breast cancer with chemotherapeutic resistance

Significance A critical challenge for chemotherapy is development of chemoresistance, but underlying molecular mechanisms remain unclear. In this study, we found that drug-resistant adriamycin-resistant human breast cancer cells possessed numerous transient receptor potential channel 5 (TrpC5) -containing extracellular vesicles (EVs) on the cell surface. Suppressing TrpC5 expression diminished the formation of EVs. Incubation of drug-sensitive recipient cells with EVs endowed recipients with drug-resistant properties. In both human samples and a mouse model of breast cancer, the expression of TrpC5 proteins was high in the tumor, and the levels of TrpC5-positive EVs were high in the circulation. These data suggest a critical role of TrpC5-containing EVs in the transfer of drug resistance. In the future, monitoring TrpC5-containing EVs in the circulation could potentially be used to predict the clinical outcome of chemotherapy. A critical challenge for chemotherapy is the development of chemoresistance in breast cancer. However, the underlying mechanisms and validated predictors remain unclear. Extracellular vesicles (EVs) have gained attention as potential means for cancer cells to share intracellular contents. In adriamycin-resistant human breast cancer cells (MCF-7/ADM), we analyzed the role of transient receptor potential channel 5 (TrpC5) in EV formation and transfer as well as the diagnostic implications. Up-regulated TrpC5, accumulated in EVs, is responsible for EV formation and trapping of adriamycin (ADM) in EVs. EV-mediated intercellular transfer of TrpC5 allowed recipient cells to acquire TrpC5, consequently stimulating multidrug efflux transporter P-glycoprotein production through a Ca2+- and activated T-cells isoform c3-mediated mechanism and thus, conferring chemoresistance on nonresistant cells. TrpC5-containing circulating EVs were detected in nude mice bearing MCF-7/ADM tumor xenografts, and the level was lower after TrpC5–siRNA treatment. In breast cancer patients who underwent chemotherapy, TrpC5 expression in the tumor was significantly higher in patients with progressive or stable disease than in patients with a partial or complete response. TrpC5-containing circulating EVs were found in peripheral blood from patients who underwent chemotherapy but not patients without chemotherapy. Taken together, we found that TrpC5-containing circulating EVs may transfer chemoresistance property to nonchemoresistant recipient cells. It may be worthwhile to further explore the potential of using TrpC5-containing EVs as a diagnostic biomarker for chemoresistant breast cancer.

MiR-489 regulates chemoresistance in breast cancer via epithelial mesenchymal transition pathway.

To investigate the role of microRNAs in the development of chemoresistance and related epithelial–mesenchymal transition (EMT), we examined the effect of miR‐489 in adriamycin (ADM)‐resistant human breast cancer cells (MCF‐7/ADM). MiR‐489 was significantly suppressed in MCF‐7/ADM cells compared with chemosensitive parental control MCF‐7/WT cells. Forced‐expression of miR‐489 reversed chemoresistance. Furthermore, Smad3 was identified as the target of miR‐489 and is highly expressed in MCF‐7/ADM cells. Forced expression of miR‐489 both inhibited Smad3 expression and Smad3 related EMT properties. Finally, the interactions between Smad3, miR‐489 and EMT were confirmed in chemoresistant tumor xenografts and clinical samples, indicating their potential implication for treatment of chemoresistance.

Acquisition of paclitaxel resistance via PI3K‑dependent epithelial‑mesenchymal transition in A2780 human ovarian cancer cells.

Epithelial ovarian cancer is a major cause of mortality among women with gynecological malignancies. Paclitaxel is commonly used for chemotherapy of ovarian cancer, yet its efficacy is limited by chemoresistance. Generally, drug resistance is associated with acquisition of the epithelial-mesenchymal transition (EMT) in cancer. The aim of the present study was to determine whether the EMT is involved in acquired resistance to paclitaxel in A2780 human ovarian cancer cells. Using the paclitaxel-resistant A2780/PTX cell line, we examined the cellular morphology, molecular changes, migration and proliferation consistent with the EMT. Furthermore, we found that inhibition of phosphatidylinositol 3-kinase (PI3K) activity reduced the proliferation and migration and restored their sensitivity to paclitaxel. Our study provides new insights into EMT-like phenotypic changes that are linked to paclitaxel resistance in A2780 cells. We believe that inhibition of the PI3K signaling pathway could provide a novel therapeutic approach to overcome chemoresistance and prevent metastasis during paclitaxel chemotherapy.

Inhibition of Transient Receptor Potential Channel 5 Reverses 5-Fluorouracil Resistance in Human Colorectal Cancer Cells

Background: Resistance to 5-fluorouracil leads to the failure of chemotherapy for colorectal cancer. Results: Suppressing TrpC5 expression decreased nuclear β-catenin accumulation, reduced the induction of ABCB1, and reversed 5-fluorouracil resistance. Conclusion: TrpC5 is essential in ABCB1 induction and drug resistance in human colorectal cancer cells. Significance: These findings may help develop a novel target for overcoming resistance to chemotherapy in colorectal cancer. 5-Fluorouracil (5-Fu) is commonly used in the chemotherapy of colorectal cancer (CRC), but resistance to 5-Fu occurs in most cases, allowing cancer progression. Suppressing ABCB1 (ATP-binding cassette, subfamily B, member 1), which is a pump overproduced in cancer cells to export cytotoxic drugs, is an attractive strategy to overcome drug resistance. In the present study, transient receptor potential channel TrpC5 was found to be overproduced at the mRNA and protein levels together with ABCB1 in 5-Fu-resistant human CRC HCT-8 (HCT-8/5-Fu) and LoVo (LoVo/5-Fu) cells. More nuclear-stabilized β-catenin accumulation was found in HCT-8/5-Fu and LoVo/5-Fu cells than in HCT-8 and LoVo cells. Suppressing TrpC5 expression with TrpC5-specific siRNA inhibited the canonical Wnt/β-catenin signal pathway, reduced the induction of ABCB1, weakened the ABCB1 efflux pump, and caused a remarkable reversal of 5-Fu resistance in HCT-8/5-Fu and LoVo/5-Fu cells. On the contrary, enforcing TrpC5 expression resulted in an activated Wnt/β-catenin signal pathway and up-regulation of ABCB1. Taken together, we demonstrated an essential role of TrpC5 in ABCB1 induction and drug resistance in human CRC cells via promoting nuclear β-catenin accumulation.

Enhancement of vascular endothelial growth factor release in long-term drug-treated breast cancer via transient receptor potential channel 5-Ca2+-hypoxia-inducible factor 1α pathway

Chemotherapy targeting anti-angiogenesis in tumors may have insufficient efficacy, but little is known about the underlying mechanisms. Here, we showed that the Ca(2+)-permeable channel, TrpC5, is highly expressed in human breast cancer after long-term chemotherapy drug-treatment. It mediates downstream hypoxia-inducible factor 1α accumulation in the nucleus, and then activates the transcription of vascular endothelial growth factor which promotes tumor angiogenesis, leading to a poor chemotherapeutic outcome. We verified this mechanism at both the cellular and xenograft levels. Moreover, in samples from patients, high TrpC5 expression was correlated with enhanced tumor vasculature after chemotherapy. Taken together, our research demonstrated the essential role of TrpC5 in tumor angiogenesis when facing the challenge of chemotherapy and presents a new potential target for overcoming the high vasculature of human breast cancer after chemotherapy.

A2780 human ovarian cancer cells with acquired paclitaxel resistance display cancer stem cell properties

The use of chemotherapy to treat cancer is effective, but chemoresistance reduces this efficacy. Chemotherapy resistance involves several mechanisms, including the cancer stem cell (CSC) concept. The aim of the present study was to assess whether paclitaxel-resistant epithelial ovarian carcinoma is capable of generating cells with CSC-like properties. Using the paclitaxel-resistant A2780/PTX cell line, it was demonstrated that high aldehyde dehydrogenase 1 (ALDH1) activity identifies CSCs from diverse sources. Furthermore, the A2780/PTX cells had a strong ability to form colonies in soft agar assays. Notably, it was demonstrated that the inhibition of the PI3K signaling pathway abolished colony formation. These data suggest that there is a link between paclitaxel resistance and CSC enrichment. It is possible that therapeutic benefits, such as the restoration of chemosensitivity or the suppression of tumorigenicity, may be enabled by gaining further insights into the mechanisms underlying chemoresistance and the generation of CSCs.

The co-stimulatory molecule B7-H3 promotes the epithelial-mesenchymal transition in colorectal cancer

B7-H3, first recognized as a co-stimulatory molecule, is abnormally expressed in cancer tissues and is associated with cancer metastasis and a poor prognosis. However, as an initial event of metastasis, the relationship between the Epithelial–Mesenchymal Transition (EMT) in cancer cells and B7-H3 has still not been investigated. In this study, we first analyzed B7-H3 expression by immunohistochemistry in colorectal cancer tissues. B7-H3 was expressed in the cancer cell membrane and was associated with the T stage of colorectal cancer; it also showed a positive correlation with MMP2 and MMP9 expression in cancer tissues. Over-expression of B7-H3 in SW480 cells allowed cancer cells to invade and metastasize more than the control cells, whereas invasion and metastasis capabilities were decreased after B7-H3 was knocked down in Caco-2 cells. We further showed that B7-H3 down-regulated the expression of E-cadherin and β-catenin and up-regulated N-cadherin and Vimentin expression, implying that B7-H3 promoted the EMT in colorectal cancer cells. We also checked another character of the EMT, the stemness of cancer cells. CD133, CD44 and Oct4 were significantly elevated after the SW480 cells were transfected with B7-H3 and reduced in Caco-2 cells after B7-H3 was inhibited. In subsequent studies, we proved that B7-H3 upregulated the expression of Smad1 via PI3K-Akt. In conclusion, B7-H3 promotes the EMT in colorectal cancer cells by activating the PI3K-Akt pathway and upregulating the expression of Smad1.

UCH-L1-containing exosomes mediate chemotherapeutic resistance transfer in breast cancer

Chemotherapy resistance has become a serious challenge in the treatment of breast cancer. Previous studies showed cells can transfer proteins, including those responsible for drug resistance to adjacent cells via exosomes.

Increasing circulating exosomes-carrying TRPC5 predicts chemoresistance in metastatic breast cancer patients.

Chemoresistance, the major obstacle in breast cancer chemotherapy, results in unnecessary chemotherapy and wasting of medical resources. No feasible method has been available to predict chemoresistance before chemotherapy. In our previous study, elevated expression of transient receptor potential channel TRPC5 was found to be an essential element for chemoresistance in breast cancer cells, and it was determined that it could be transferred to chemosensitive breast cancer cells through releasing extracellular vesicles (EV) containing TRPC5 from chemoresistant cells, resulting in acquired chemoresistance. Exosomes, a type of EV, are secreted membrane‐enclosed vesicles of 50–150‐nm diameter. In this study we found that circulating exosomes in peripheral blood from breast cancer patients carried TRPC5. In the present study, circulating exosome‐carrying TRPC5 (cirExo‐TRPC5) level was significantly correlated with TRPC5 expression level in breast cancer tissues and tumor response to chemotherapy. Furthermore, increased cirExo‐TRPC5 level after chemotherapy preceded progressive disease (PD) based on imaging examination and strongly predicted acquired chemoresistance. Taken together, our study demonstrated that cirExo‐TRPC5 might act as a noninvasive chemoresistance marker and might serve as an adjuvant to the current imaging examination‐based chemoresistance.

Preoperative plasma fibrinogen, but not D-dimer might represent a prognostic factor in non-metastatic colorectal cancer: A prospective cohort study

BACKGROUNDS Cancer progression has been associated with host hemostasis system. Whether preoperative plasma hemostasis factors can predict survival in colorectal cancer is quite intriguing. METHODS We conducted a prospective cohort study to validate the prognostic significance of three hemostasis parameters - fibrinogen, fibrin degradation products (FDPs) and D-dimer - in non-metastatic colorectal cancer patients treated with curative resection. RESULTS All three parameters were positively correlated with C reactive protein (CRP) levels and Glasgow Prognostic scores (GPS). In univariate cox hazards regression model, as continuous variables, both fibrinogen (HR: 1.07, 95%CI: 1.01-1.13) and FDPs (HR: 1.17, 95%CI: 1.05-1.31) were prognostic, while D-dimer levels were not. Patients with hyperfibrinogenemia had a 2.12-fold increased mortality risk compared with patients without hyperfibrinogenemia. Patients with positive FDPs had a 3.68-fold increased mortality risk compared with patients with negative FDPs. In multivariate models, hyperfibrinogenemia was prognostic (HR: 3.39, 95%CI: 1.34-8.67) in patients with normal GPS scores. CONCLUSIONS Preoperative fibrinogen levels appeared as an independent mortality risk factor in non-metastatic colorectal cancer patients with normal GPS scores. Fibrinogen could be a reliable marker to identify high risk patients for those without systematic inflammation responses.